Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Platelet activation and blood extracellular vesicles: The influence of venepuncture and short blood storage.

Author(s): Marić I, Žiberna K, Kolenc A, Maličev E

Publication: Blood Cells Mol Dis, 2024, Vol. 106, Page 102842

PubMed ID: 38492545 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of volunteer characteristics (age, gender, smoking status, and fasting status), needle gauge (16- versus 21-), and delayed plasma separation (up to 4 h at room temperature) on platelet activation and EV endpoints by comparing the percentage of activated platelets relative to total platelets (CD62p+/CD61+) and the concentration, size, and surface marker MFI among EVs isolated from the plasma of twenty healthy volunteers.  Unless otherwise specified, blood was collected using a 21-gauge needle from the cubital vein of 17 healthy men (24-62 years of age) and three healthy women (24-62 years of age); donors included 18 non-smokers and 2 smokers; 17 non-fasting and 3 fasting individuals.  Blood was collected into EDTA Monovette tubes and was mixed by inverting the tube six times before separation of platelet-rich plasma (PRP) by centrifugation at 120 x g for 7 min. PDP was isolated from PRP by two centrifugations at 2500 x g for 15 min at room temperature and stored at -80°C until analysis. Platelet activation was measured by the ratio of activated (CD62p+) to total (CD61+) platelets by flow cytometry. EVs were isolated from PDP by centrifugation at 10,000 × g for 20 min at 4°C, mixing the resultant supernatant with sucrose and phosphate-buffered saline (PBS), and ultracentrifugation at 100,000 × g for 135 min at 4°C. EVs were resuspended in PBS and stored at -80°C. Evs were assayed by nanoparticle tracking and flow cytometry for 37 surface markers, of which 30 were detected. For needle gauge comparisons, blood was collected from 10 men using a 21-gauge needle followed by a collection of a subsequent sample using a 16-gauge needle. For delayed processing comparisons, blood from seven men and three women was mixed by inverting the tubes six times and storing the pool sample at room temperature for 0, 1, 2, 3, or 4 h before separation of platelet rich plasma. Correlation coefficients were not included for most correlation analyses.

   Summary of Findings:

   Plasma from the seventeen male donors had a significantly higher concentration of EVs than plasma from the three female donors (5.19 versus 2.62, P=0.42), but EV concentration was not affected by volunteer age, smoking status (18 non-smokers, 2 smokers), or fasting status (17 non-fasting, 3 fasting)). EV size was correlated with EV concentration (P<0.001), but was not affected by volunteer gender, age, smoking status, or fasting status. Platelet activation was highly variable between volunteers (CV 68%), but was not affected by volunteer gender, age, smoking status, or fasting status. Platelet activation was also not correlated with EV concentration or size.  Except for a higher mean fluorescent intensity (MFI) of HLA-DRDQDP in EVs from men in comparison to those from women (33.4 vs. 15.1, P=0.019), there were no effects of volunteer gender, age, smoking status, or fasting status on the MFI of the thirty EV surface markers investigated, but variability was high (CV 49-364%).  EV concentration was lower when blood was collected using a 16-gauge rather than 21-gauge needle (3.07 versus 4.62, P=0.02), but EV concentration, platelet activation, and EV surface markers were comparable between specimens collected with different needle gauges.  Platelet activation increased with the duration of room temperature storage prior to plasma separation (P=0.01), but the duration of blood storage did not affect the concentration, size, or MFI of surface markers of EVs in plasma.  EV concentration was negatively correlated with the MFI of some markers, including CD49e (P=0.036), CD40 (P 0.044), CD41b (P=0.025), CD62p (P=0.037), CD146 (P=0.030), and CD31 (P = 0.024). EV size was modestly correlated with the MFI of CD62p (P=0.001, R2=0.491) and CD40 (P=0.001, R2=0.473).

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Morphology	Light scattering
   Cell count/volume	Light scattering
   Protein	Flow cytometry
   Cell count/volume	Flow cytometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Fasting Non-fasting Smoker Non-smoker
   Preaquisition	Patient age	24-62 years of age
   Preaquisition	Patient gender	Female Male
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Biospecimen Acquisition	Time of biospecimen collection	7-8 AM fasting 7-8 AM non-fasting
   Storage	Time at room temperature	0 h 1 h 2 h 3 h 4 h
   Biospecimen Acquisition	Needle gauge	16-gauge 21-gauge

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